Nitric oxide (NO) is an important signaling molecule involved in the regulation of many physiological and pathological functions of the vascular endothelium. The objectives of this proposal are to investigate the mechanisms regulating endothelial NO synthase (eNOS) activity in intact microvessels under inflammatory conditions and their direct relationship with microvessel permeability. The hypothesis to be tested is that Ca2+/calmodulin (CaM) and heat shock protein 90 (hsp90) play antagonistic roles to caveolin-1 in the regulation of eNOS activity and microvessel permeability through competing protein-protein interactions in intact microvessels, and agonist-stimulated NO production is critical for increases in permeability under inflammatory conditions. Individually perfused intact venular microvessels in rat mesentery will be used to perform the proposed studies. The unique advantage of using single vessel perfusion technique over other whole animal approaches for this specific study is that it allows the NO effect on blood cell/endothelium interactions and on the changes in hemodynamics in the vasculature to be distinguished from its role in the regulation of microvessel permeability. Agonist-stimulated NO production will be quantitatively measured at levels of individual endothelial cells in intact microvessels with temporal and spatial resolution using fluorescence imaging. The role of Ca2+/CaM in the regulation of eNOS activity will be investigated by measuring corresponding changes in NO production when agonist-induced Ca2+ influx is modified following changes in electrochemical driving force for Ca2+ entry in endothelial cells. Our newly developed methods also allow a targeted regulatory protein for eNOS such as caveolin-1 or hsp90 to be internalized or genetically expressed in endothelial cel|s that form intact microvessels. In this proposal we combined our newly developed molecular approaches with our previously established techniques to investigate the mechanisms regulating eNOS activity in intact microvessels. Because of the nonuniformity nature of leaky site formation along the microvessel walls during acute inflammation, the spatial heterogeneity correlation between endothelial [Ca2+]i and NO production will be quantitatively evaluated at cellular levels in intact microvessels. The proposed research will provide new information that bridges the studies using whole animal, organ, or vascular beds and studies with purified proteins or endothelial cells in culture.